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S R U T H Y K . S
1 1 - B V P - 2 1 6
VPB 321
DNA LIBRARIES
DNA LIBRARY
The term "library" can refer to a population of
organisms, each of which carries a DNA molecule
inserted into ...
Genomic libraries
cDNA libraries
Gene
library
(made from genomic DNA)
(made from cDNA- copy of mRNA)
For the construction of DNA library
Size of the gene
Capacity of the vector
Molecular tools
Vectors
Vector type Max. Insert size
cloned DNA
(kb)
Approx.no: of cloned
DNA required in a
library
Plasmid 20 >10^5
Lambda phage ...
Genomic DNA library: Contains the whole
genome of an organism
Genome size is expressed in terms of number of base
pairs.
S...
STEPS
1) Purification of genomic DNA
PROKARYOTES
EUKARYOTES
•Genomic libraries of prokaryotes are
easier to make and conta...
2) Cleaving the genome into smaller fragments
by restriction endonucleases.
• Parial digestion is used to getting longer D...
3) In cooperation of these fragments into a
suitable vector.
4) Introduction of vector into a suitable host
like bacterium(Transformation)
 Multiplication
 Screening
Screening:
The process of identifying one particular clone
containing the gene of interest from among
the very large numbe...
Expression screening
Eg:
Finding the gene for alanine production
Grow in alanine deficit medium
Then they are labelled in ...
Hybridisation technique
cDNA Library
No cDNA library was made from prokaryotic
mRNA…..???
cDNA libraries are very useful for eukaryotic gene
analy...
mRNA isolation, purification
Check the RNA integrity
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector
mRNA isolation
Most eukaryotic mRNAs are polyadenylated
at their 3’ ends
• oligo (dT) can be bound to the poly(A) tail and...
Check the mRNA integrity
Make sure that the mRNA is not degraded.
•Translating the mRNA
•Analysis the mRNAs by gel
elctrop...
Synthesis of cDNA
First stand synthesis:
Reverse transcriptase ,primer( oligo(dT) or
hexanucleotides) and dNTPs.
Second st...
5’ mRNA AAAAA-3’
HO-TTTTTP-5’
5’
Reverse transcriptase
Four dNTPs
AAAAA-3’
TTTTTP-5’
mRNA
mRNA
cDNA
cDNA
cDNA
Duplex cDNA
...
5’-pGGGG
3’-CCCCCCC
HO-CCGAATTCGGGGGG
3’-GGCTTAAGCCCCCC
5’-pAATTCGGGGGG
TTTTTGGCTTAAGCC-OH
CCGAATTCGG-3’
3’-CCCC
3’-CCCCCC...
Treatment of cDNA ends
Blunt and ligation of large fragment is not efficient, so we have to
use special acid linkers to cr...
Ligation to vector
Any vectors with an EcoRI site would suitable
for cloning the cDNA.
Dephosphorylate the vector with alk...
Uses of cDNA library
Used when reproducing eukaryotic genomes as
the amount of information is reduced to remove the
large ...
DNA Libraries PPT
DNA Libraries PPT
DNA Libraries PPT
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DNA Libraries PPT

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DNA Libraries are collection of fragments of DNA cloned to a vector so that researchers can easily identify and isolate a particular gene of interest for future use.

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DNA Libraries PPT

  1. 1. S R U T H Y K . S 1 1 - B V P - 2 1 6 VPB 321 DNA LIBRARIES
  2. 2. DNA LIBRARY The term "library" can refer to a population of organisms, each of which carries a DNA molecule inserted into a cloning vector, or alternatively to the collection of all of the cloned vector molecules. Collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study.
  3. 3. Genomic libraries cDNA libraries Gene library (made from genomic DNA) (made from cDNA- copy of mRNA)
  4. 4. For the construction of DNA library Size of the gene Capacity of the vector Molecular tools Vectors
  5. 5. Vector type Max. Insert size cloned DNA (kb) Approx.no: of cloned DNA required in a library Plasmid 20 >10^5 Lambda phage 20 5 x10^5 Cosmid 45 2 x 10^5 BAC >500 5 x 10^4 YAC 1Mb 1o^5
  6. 6. Genomic DNA library: Contains the whole genome of an organism Genome size is expressed in terms of number of base pairs. Smallest genomic size is…………..???? For human the genomic size is………….????
  7. 7. STEPS 1) Purification of genomic DNA PROKARYOTES EUKARYOTES •Genomic libraries of prokaryotes are easier to make and contain all the genome sequences.
  8. 8. 2) Cleaving the genome into smaller fragments by restriction endonucleases. • Parial digestion is used to getting longer DNA sequences.
  9. 9. 3) In cooperation of these fragments into a suitable vector.
  10. 10. 4) Introduction of vector into a suitable host like bacterium(Transformation)
  11. 11.  Multiplication  Screening
  12. 12. Screening: The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library .  Expression screening  Hybridisation technique  PCR
  13. 13. Expression screening Eg: Finding the gene for alanine production Grow in alanine deficit medium Then they are labelled in the petri plate indicates that gene for alanine production is stored in bacteria.
  14. 14. Hybridisation technique
  15. 15. cDNA Library No cDNA library was made from prokaryotic mRNA…..??? cDNA libraries are very useful for eukaryotic gene analysis.
  16. 16. mRNA isolation, purification Check the RNA integrity Synthesis of cDNA Treatment of cDNA ends Ligation to vector
  17. 17. mRNA isolation Most eukaryotic mRNAs are polyadenylated at their 3’ ends • oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA. AAAAAAAAAAn5’ cap
  18. 18. Check the mRNA integrity Make sure that the mRNA is not degraded. •Translating the mRNA •Analysis the mRNAs by gel elctrophoresis
  19. 19. Synthesis of cDNA First stand synthesis: Reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs. Second strand synthesis: Best way of making full-length cdna is to ‘tail’ the 3’-end of the first strand and then use a complementary primer to make the second.
  20. 20. 5’ mRNA AAAAA-3’ HO-TTTTTP-5’ 5’ Reverse transcriptase Four dNTPs AAAAA-3’ TTTTTP-5’ mRNA mRNA cDNA cDNA cDNA Duplex cDNA AAAAA-3’ TTTTTP-5’ TTTTTP-5’ 3’ 3’-CCCCCCC Terminal transferase dCTP Alkali (hydrolyaes RNA) Purify DNA oligo(dG) Klenow polymerase or reverse Transcriotase Four dNTPs 5’-pGGGG-OH 5’ 3’-CCCCCCC 5’-pGGGG 3’-CCCCCCC TTTTTP-5’ -3’ Angelia 09 22
  21. 21. 5’-pGGGG 3’-CCCCCCC HO-CCGAATTCGGGGGG 3’-GGCTTAAGCCCCCC 5’-pAATTCGGGGGG TTTTTGGCTTAAGCC-OH CCGAATTCGG-3’ 3’-CCCC 3’-CCCCCCC 3’-CCC 5’-pGGGG 5’-pGGGG TTTTTp-5’ -3’ TTTTTp-5’ TTTTTp-5’ -3’ -3’ TTTTTGGCTTAAp-5’ HO-CCG/AATTCGG-3’ 3’-GGCTTAA/GCC-OH CCG-3’ Duplex cDNA Single strand-specific nuclease Klenow polymerase treat with E.coRI methylase Add E.colRI linkers using T4 DNA ligase E.colRI digestion Ligate to vector and transfom Fig2.1 Second strand synthesis23
  22. 22. Treatment of cDNA ends Blunt and ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning. Move protruding 3’-ends (strand-special nuclease) Fill in missing 3’ nucleotide Ligate the blunt-end and linkers(T4 DNA ligase) Restriction enzyme digestion (E.coRI ) Tailing with terminal transferase or using adaptor molecules 24
  23. 23. Ligation to vector Any vectors with an EcoRI site would suitable for cloning the cDNA. Dephosphorylate the vector with alkaline phosphatase Ligate vector and cDNA with T4 DNA ligase (plasmid or λ phage vector) 25
  24. 24. Uses of cDNA library Used when reproducing eukaryotic genomes as the amount of information is reduced to remove the large number of non-coding regions from the library. To express eukaryotic genes in prokaryotes. Useful for subsequently isolating the gene that codes for that mRNA.

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